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Journal of Biochemistry Advance Access originally published online on January 22, 2008
Journal of Biochemistry 2008 143(4):455-465; doi:10.1093/jb/mvn005
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© 2008 The Japanese Biochemical Society.

Analysis of Origin Recognition Complex in Saccharomyces cerevisiae by Use of Degron Mutants

Masaki Makise1,{dagger}, Nanako Matsui1,{dagger}, Fumiko Yamairi1,{dagger}, Naoko Takahashi2, Masaya Takehara1, Teita Asano1 and Tohru Mizushima1,*

1Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973; and 2Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan

*To whom correspondence should be addressed. Tel: +81 96 371 4323, Fax: +81 96 371 4323, E-mail: mizu{at}gpo.kumamoto-u.ac.jp

Received November 22, 2007; Accepted December 7, 2007


  Abstract

Origin recognition complex (ORC), a six-protein complex (Orc1p-Orc6p), may deeply involve in initiation of chromosomal DNA replication. However, since most temperature-sensitive orc mutants of Saccharomyces cerevisiae show the accumulation of cells with nearly 2C DNA content, the exact stage at which ORC acts is not fully understood. In this study, we constructed a heat-inducible degron mutant for each ORC subunit. As well as each targeted subunit, other subunits of ORC were also rapidly degraded under non-permissive conditions. In the orc5 degron mutant, incubation under the non-permissive conditions caused accumulation of cells with nearly 2C DNA content, and phosphorylation of Rad53p. When Orc5p (ORC) is depleted, this inhibits G1/S transition and formation of a pre-replicative complex (pre-RC). For pre-RC to form, and G1/S transition to proceed, Orc5p (ORC) must be present in late G1, rather than early G1, or G2/M. Block and release experiments revealed that Orc5p (ORC) is not necessary for S and G2/M phase progression. We therefore propose that ORC is necessary for the G1/S transition and pre-RC formation, and accumulation of cells with nearly 2C DNA content seen in various orc mutants is due to inefficient pre-RC formation, and/or induction of checkpoint systems.

Key Words: DNA replication, heat inducible degron mutant, ORC, Orc5p, pre-replicative complex

Abbreviations: {alpha}-factor, {alpha} mating factor; DHFR, dihydrofolate reductase; FACS, fluorescence-activated cell sorter; HA, haemagglutinin; HU, hydroxyurea; MCM, mini-chromosome maintenance complex of proteins; ORC, origin recognition complex; pre-RC, pre-replication complex

{dagger}These authors contributed equally to this work.


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